Murine Model Of Acute Myocarditis Research Articles

Immunotherapy-related cardiovascular toxicity: Development of a preclinical murine model of acute myocarditis

Immunotherapies represent a major advance in oncology and an increasing number of patients will benefit from them. However, activation of the immune system can lead to severe myocarditis with a fatality rate up to 50%. The pathophysiological mechanisms of myocarditis induced by anti-PD-1 and anti-CTLA-4, the most commonly used immune checkpoint inhibitor (ICIs), are poorly understood and require models to study this cardiotoxicity. PD-1 and CTLA-4 KO mouse models do show cardiomyopathy but still do not reflect the immuno-induced myocarditis observed in patients. Our objective is to develop a preclinical mouse model to characterize in vivo the pathophysiological mechanisms of ICIs-induced myocarditis. We used a syngeneic model with adult BALB/c mice grafted with murine melanoma cells. Ten days after graft, mice were treated every 2 days with peritoneal injection of a combination of anti-PD-1 (6.5 mg/kg) and anti-CTLA-4 (20 mg/kg) monoclonal antibodies. A group of ICIs-treated mice with melanoma also received diphtheria toxin to amplify the cardiotoxic response, by depletion of regulatory T cells. To follow-up the cardiac function, an ECG was recorded before each ICIs injection and 1 week after the 3rd injection before euthanasia. A submaxillary blood sample was taken before and after ICIs injection. To investigate inflammation, hearts were dissected for transcriptomic analysis and immunostaining. All transplanted mice developed a palpable melanocytic tumor. They maintained a stable clinical condition (no weight loss or signs of pain). ECG analysis showed some arrhythmias and numerous extrasystoles. Analyses are currently underway to determine the characteristics of the different intervals in each of the groups tested and to verify the significance of the results. qPCR analyzes are in progress to verify the inflammatory state of the myocardium. We are looking for changes in the expression of genes of the JAK/STAT pathway and of the NLRP3 inflammasome, including the GBP5 , GBP6 , and CCDC175 , CXCL9 , CXCL11 genes which have been described in cardiac biopsies from patients. This model of in vivo cardiotoxicity is very important to unravel the mechanisms that lead to immuno-induced myocarditis. The results obtained will be integrated with data from patients followed at the Cardio-Oncology center in Marseille and those from the in vitro hiPSC model in order to better understand the pathology and identify prognosis markers.

Murine model of acute myocarditis and cerebral cortical neuron edema induced by coxsackievirus B4.

Globally, coxsackievirus B4 (CV-B4) has been continuously isolated and evidence suggests an association with the development of pancreatitis and type I diabetes. In addition, CV-B4 is also associated with myocarditis and severe central nervous system (CNS) complications, which remain poorly studied and understood. In the present study, we established an Institute for Cancer Research (ICR) mouse model of CV-B4 infection and examined whether CV-B4 infection resulted in a predisposition to myocarditis and CNS infection. We found high survival in both the treatment and control group, with no significant differences in clinical outcomes observed. However, pathological lesions were evident in both brain and heart tissue of the CV-B4-infected mice. In addition, high viral loads were found in the neural and cardiac tissues as early as 2 days post infection. Expressions of IFN-γ and IL-6 in sera were significantly higher in CV-B4-infected mice compared to uninfected negative controls, suggesting the involvement of these cytokines in the development of histopathological lesions. Our murine model successfully reproduced the acute myocarditis and cerebral cortical neuron edema induced by CV-B4, and may be useful for the evaluation of vaccine candidates and potential antivirals against CV-B4 infection.

Oral Administration of Candesartan Improves the Survival of Mice with Viral Myocarditis through Modification of Cardiac Adiponectin Expression

We examined the effects of the angiotensin II receptor type 1 blocker candesartan on myocarditis injury in a murine model of acute myocarditis. We hypothesized that candesartan improves cardiac damage by inducing cardiac expression of adiponectin. We examined changes in heart failure caused by myocarditis in mice by candesartan based on induction of cardiac adiponectin expression. We intraperitoneally injected encephalomyocarditis virus in C3H mice, then orally administered candesartan (10 mg/kg/day) or vehicle (control). The 7 day survival rate was 18% in the control group, but 60% in the candesartan group. The heart weight/body weight ratio in the candesartan group was significantly lower than in the control group. Circulating adiponectin concentrations on day 7 were significantly higher in the candesartan group compared with the control group (7.91 +/- 0.61 vs. 6.04 +/- 2.26 microg/ml, P < 0.05). Comparative expression of cardiac adiponectin mRNA in the candesartan group was significantly higher than in the control group on day 7 (55.4 +/- 41.3 vs. 5.3 +/- 7.7, P < 0.05). Immunohistochemical staining and in situ hybridization showed that cardiac expression of adiponectin protein and mRNA was present in the candesartan group on day 7. Oral administration of candesartan improves survival and decreases myocardial damage in mice with viral myocarditis and induces expression of cardiac adiponectin. The induction of adiponectin might provide cardioprotective effects against acute heart failure due to viral myocarditis.

Expressional Patterns of Cytokines in a Murine Model of Acute Myocarditis: Early Expression of Cardiotrophin-1

To determine the role of cytokines in acute myocarditis, we examined expressional patterns of cardiotrophin-1 (CT-1), TNF-α, and IL-1α in a murine model of acute myocarditis. Ten-day-old Institute of Cancer Research mice were injected with Coxsackievirus B3 and killed on Days 1, 2, 3, 4, 5, 7, 10, 14, and 28 of injection. TNF-α and IL-1α expressions were investigated on histological sections from each heart, and mRNA expression of TNF-α, IL-1α, and CT-1 in the heart was examined by reverse transcription-polymerase chain reaction and RNase protection assay. To determine myocardial regeneration, cardiomyocytic DNA synthesis was investigated using bromodeoxyuridine on Days 3, 5, 7, and 10, and the labeling index was calculated in each heart. Age-matched uninfected mice were used as controls. TNFα and IL-1α expression was first detected in the cardiomyocytes on Day 3 and reached the maximum level on Day 7, when inflammatory changes were most prominent. Although an increased expression of TNFα and IL-1α mRNA was also detected on Day 3, CT-1 mRNA expression was distinctly augmented on Day 2. The labeling indices in the hearts with myocarditis were significantly higher than in those of the controls in all of the time points examined. CT-1 expression preceded TNF-α and IL-1α expressions and active DNA synthesis in a murine model of acute myocarditis. All CVB3-infected mice with anti-glycoprotein-130 antibody treatment died within 6 days. CT-1 may exert a protective role by modulating cytokine production and by inducing cardiomyocytic proliferation in CVB3-infected murine hearts.

Genomic regions of coxsackievirus B3 associated with cardiovirulence

The molecular basis for cardiovirulence in the coxsackievirus B3 (CVB3) genome was examined in a murine model of acute myocarditis. Infectious cDNAs representing a highly cardiovirulent coxsackievirus B3 (CVB3m) and a noncardiovirulent (CVB30) virus were used to construct infectious chimeric cDNAs. Assays of the resulting recombinant viruses for cardiovirulence in adolescent male CD-1 mice showed that the 5' nontranslated region (5' NTR) of the CVB3m genome plays the major role in determining cardiovirulence and that the genomic region encoding the capsid proteins has a minor additive effect in increasing cardiovirulence. Nucleotide sequences in the 5' NTR of CVB3m and CVB30 differ at 23 positions; 14 are located in four stemloop motifs of the secondary structure and may influence the cardiovirulent phenotype by regulating RNA or protein synthesis. A comparison of predicted amino acid sequences of capsid proteins in CVB3m and CVB30 identified two amino acids as potential candidate contributors to cardiovirulence, i.e., amino acids at positions A207 (Asn-Asp) in the puff structure of the E-F loop of VP2 and A566 (Gln-Glu) in the C terminal of VP3 at the external surface. The data from this study and published literature support the conclusion that cardiovirulence of a CVB3 can depend on several regions of the genome.

Expression of vascular cell adhesion molecule-1 in murine hearts with acute myocarditis caused by coxsackievirus B3.

Cell-mediated autoimmunity has been strongly implicated in the pathogenesis of the myocardial cell damage involved in viral myocarditis. Using a murine model of acute myocarditis caused by Coxsackievirus B3 (CVB3), perforin-expressing killer cells have been shown to infiltrate the heart, and intercellular adhesion molecular-1 (ICAM-1) together with major histocompatibility complex (MHC) antigen was induced on myocardial cells in acute viral myocarditis. To clarify the immunological mechanisms in more detail, the expression of vascular cell adhesion molecular-1 (VCAM-1) has been examined in the heart of acute myocarditis and on cultured cardiac myocytes treated with interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha). The effects of in vivo antibody treatment to VCAM-1 on myocardial damage involved in acute myocarditis were also analysed. CVB3-induced myocarditis resulted in enhanced expression of VCAM-1 on myocardial cells. VCAM-1 expression was also induced on cultured cardiac myocytes by treatment with IFN-gamma and TNF-alpha. The in vivo antibody treatment to VCAM-1 decreased the myocardial damage to some extent, but the effects were not statistically significant. These data suggest that the expression of VCAM-1 on myocardial cells may play at least a partial role in the myocardial damage involved in acute viral myocarditis.

T-cell receptor V beta gene expression in infiltrating cells in murine hearts with acute myocarditis caused by coxsackievirus B3.

In viral myocarditis, we previously reported that natural killer cells infiltrate the heart first, then activated T cells infiltrate second and play an important role in the pathogenesis of the myocardial damage. To elucidate the nature of T-cell infiltration, using a murine model of acute myocarditis caused by coxsackievirus B3, we analyzed the expression of T-cell receptor (TCR) V beta genes in infiltrating cells in the heart by polymerase chain reaction (PCR). The PCR-amplified products were confirmed by Southern blot hybridization with a C beta cDNA probe. In contrast to spleen lymphocytes, the repertoire of V beta gene transcripts in the heart was restricted. The infiltrating cells expressing V beta 10 were found in six of eight hearts of mice with acute myocarditis. The infiltrating cells expressing V beta 8 and V beta 13 were found in four of eight hearts with myocarditis, respectively. Immunoperoxidase staining of serial sections of the heart of myocarditis for TCR alpha beta chains and TCR V beta 10 confirmed that the dominant population of infiltrating T cells expressed V beta 10 gene products. The restricted usage of TCR genes by infiltrating T-cells may indicate that a specific antigen in heart with myocarditis is targeted. Our findings raise the possibility of immunotherapy with monoclonal antibodies specific for TCR V beta elements to prevent T-cell-mediated myocardial damage in viral myocarditis.

Expression of perforin in infiltrating cells in murine hearts with acute myocarditis caused by coxsackievirus B3.

Cell-mediated autoimmunity has been strongly implicated in the pathogenesis of viral myocarditis. Using a murine model of acute myocarditis caused by coxsackievirus B3, we analyzed the phenotypes and morphology of the infiltrating cells in the hearts by immunofluorescence and electron microscopy. We also examined the expression of a cytolytic factor, perforin, in the infiltrating cells by immunoperoxidase and in situ hybridization. We found that the dominant population of the infiltrating cells were asialo GM1 positive, were negative for T-cell markers, and had electron-dense cytoplasmic granules, which is consistent with a morphology of large granular lymphocytes. Perforin was found in the cytoplasmic granules of the infiltrating cells expressing perforin messenger RNA. These findings provide for the first time the direct evidence that the first wave of cell infiltration in hearts mainly consists of killer cells and strongly suggests that perforin plays, in part, an important role in myocardial cell damage involved in acute viral myocarditis. T-helper cells and cytotoxic T lymphocytes made up the second wave of cell infiltration. As we previously reported, the expression of major histocompatibility complex class I antigen on cardiac myocytes induced by the infiltrating cells, such as killer cells, may facilitate the interaction between cardiac myocytes and cytotoxic T lymphocytes, and may lead to further myocardial cell damage in a later phase.